We report the cloning and expression pattern of Ci-msxb the second Ciona intestinalis homeobox gene homologue to the Drosophila muscle segment homeobox (msh) gene. Northern blot analysis showed that transcripts appeared at gastrula stage, peaked in the early tailbud and decreased during the tailed stages. Whole mount in situ hybridization showed that the Ci-msxb expression first is detected at 110 cell-stage in the blastomeres that are precursors of different tissue (muscle, spinal cord, endodermal strand, brain, mesenchyme, pigmented cells and primordial pharynx). Transcript level declined in mesoderm cells after the completion of gastrulation, but mRNAs were still present in the folding neural plate during neurulation and in the pigmented cells. Later, at larval stage, transcripts were present around the otolith and ocellus, in a restricted part of the nervous system and in the primordial pharynx; the gene expression was conserved after metamorphosis in the juvenile.